Investigation of the regulation of carbohydrate metabolism in Arabidopsis thaliana using a genetic approach

The regulation of carbohydrate metabolism in Arabidopsis thaliana was investigated using a genetic approach. A new class of carbohydrate insensitive mutant (cai) was characterised in order to gain insight into the control of carbohydrate metabolism. Wild type seedlings germinated on media containing 100 mM sucrose and 0.1 mM nitrogen but their cotyledons did not expand and accumulated anthocyanins. After 1 week growth was arrested. The internal carbohydrate content increased accompanied by repression of photosynthetic genes and induction of chs gene expression, cai mutants germinated on agar media containing 100 mM sucrose and 0.1 mM nitrogen but their cotyledons expanded and greened. After initial characterisation of a number of the mutants, two were selected for further analysis. When germinated on a range of different carbon; nitrogen ratios cai 10 and cai 28 displayed a reduced sensitivity to the high carbohydrate and low nitrogen conditions, cai 10 also displayed a mannose insensitive (mig) phenotype compared to the post-germinative growth of wild type which was arrested by mannose. This growth arrest in the wild type on mannose correlates with phosphate sequestration, cai 10 metabolises mannose at a different rate and accumulates less hexose phosphate than the wild type when germinated on mannose, thus indicating that the mannose insensitive phenotype may be a consequence of a disruption in metabolism. Overexpression of Arabidopsis hexokinase 1 in cai 10 did not complement the cai 10 phenotype. In contrast to previous results by Jang et al., (1997), who found that plants overexpressing hexokinase were hypersensitive to sugars, our results indicate that they are less sensitive than wild type. This is not in agreement with the proposed model of hexokinase as a sugar sensor (Jang et al., 1997). Seeds of the hexokinase overexpressors germinated rapidly (within 18-20 h). The seeds also contained elevated levels of some amino acids, smaller lipid bodies and less lipid than the wild type. It is proposed that hexokinase overexpression increases glucose-6-phosphate concentration which activates phosphoenolpyravate carboxylase (PEPCase) and in so doing diverts carbon from lipid biosynthesis to amino acid synthesis

Crassulacean acid metabolism guard cell anion channel activity follows transcript abundance and is suppressed by apoplastic malate

* Plants utilizing crassulacean acid metabolism (CAM) concentrate CO2 around RuBisCO while reducing transpirational water loss associated with photosynthesis. Unlike stomata of C3 and C4 species, CAM stomata open at night for the mesophyll to fix CO2 into malate (Mal) and store it in the vacuole. CAM plants decarboxylate Mal in the light, generating high CO2 concentrations within the leaf behind closed stomata for refixation by RuBisCO. * CO2 may contribute to stomatal closure but additional mechanisms, plausibly including Mal activation of anion channels, ensure closure in the light. * In the CAM species Kalanchoë fedtschenkoi, we found that guard cell anion channel activity, recorded under voltage clamp, follows KfSLAC1 and KfALMT12 transcript abundance, declining to near‐zero by the end of the light period. Unexpectedly, however, we found that extracellular Mal inhibited the anion current of Kalanchoë guard cells, both in wild‐type and RNAi mutants with impaired Mal metabolism. * We conclude that the diurnal cycle of anion channel gene transcription, rather than the physiological signal of Mal release, is a key factor in the inverted CAM stomatal cycle

Phosphorylation of Phosphoenolpyruvate Carboxylase Is Essential for Maximal and Sustained Dark CO2 Fixation and Core Circadian Clock Operation in the Obligate Crassulacean Acid Metabolism Species Kalanchoe fedtschenkoi

Phosphoenolpyruvate carboxylase (PPC; EC 4.1.1.31) catalyzes primary nocturnal CO2 fixation in Crassulacean acid metabolism (CAM) species. CAM PPC is regulated posttranslationally by a circadian clock-controlled protein kinase called phosphoenolpyruvate carboxylase kinase (PPCK). PPCK phosphorylates PPC during the dark period, reducing its sensitivity to feedback inhibition by malate and thus enhancing nocturnal CO2 fixation to stored malate. Here, we report the generation and characterization of transgenic RNAi lines of the obligate CAM species Kalanchoë fedtschenkoi with reduced levels of KfPPCK1 transcripts. Plants with reduced or no detectable dark phosphorylation of PPC displayed up to a 66% reduction in total dark period CO2 fixation. These perturbations paralleled reduced malate accumulation at dawn and decreased nocturnal starch turnover. Loss of oscillations in the transcript abundance of KfPPCK1 was accompanied by a loss of oscillations in the transcript abundance of many core circadian clock genes, suggesting that perturbing the only known link between CAM and the circadian clock feeds back to perturb the central circadian clock itself. This work shows that clock control of KfPPCK1 prolongs the activity of PPC throughout the dark period in K. fedtschenkoi, optimizing CAM-associated dark CO2 fixation, malate accumulation, CAM productivity, and core circadian clock robustness

Kalanchoe PPC1 Is Essential for Crassulacean Acid Metabolism and the Regulation of Core Circadian Clock and Guard Cell Signaling Genes([CC-BY])

Unlike C3 plants, Crassulacean acid metabolism (CAM) plants fix CO2 in the dark using phosphoenolpyruvate carboxylase (PPC; EC 4.1.1.31). PPC combines phosphoenolpyruvate with CO2 (as HCO3−), forming oxaloacetate. The oxaloacetate is converted to malate, leading to malic acid accumulation in the vacuole, which peaks at dawn. During the light period, malate decarboxylation concentrates CO2 around Rubisco for secondary fixation. CAM mutants lacking PPC have not been described. Here, we employed RNA interference to silence the CAM isogene PPC1 in Kalanchoë laxiflora. Line rPPC1-B lacked PPC1 transcripts, PPC activity, dark period CO2 fixation, and nocturnal malate accumulation. Light period stomatal closure was also perturbed, and the plants displayed reduced but detectable dark period stomatal conductance and arrhythmia of the CAM CO2 fixation circadian rhythm under constant light and temperature free-running conditions. By contrast, the rhythm of delayed fluorescence was enhanced in plants lacking PPC1. Furthermore, a subset of gene transcripts within the central circadian oscillator was upregulated and oscillated robustly in this line. The regulation of guard cell genes involved in controlling stomatal movements was also perturbed in rPPC1-B. These findings provide direct evidence that the regulatory patterns of key guard cell signaling genes are linked with the characteristic inverse pattern of stomatal opening and closing during CAM

Silencing PHOSPHOENOLPYRUVATE CARBOXYLASE1 in the Obligate Crassulacean Acid Metabolism Species Kalanchoë laxiflora causes Reversion to C3-like Metabolism and Amplifies Rhythmicity in a Subset of Core Circadian Clock Genes

ABSTRACT Unlike C 3 plants, Crassulacean acid metabolism (CAM) plants fix CO 2 in the dark using phosphoenolpyruvate carboxylase (PPC; EC 4.1.1.31). PPC combines PEP with CO 2 (as HCO 3 − ), forming oxaloacetate that is rapidly converted to malate, leading to vacuolar malic acid accumulation that peaks phased to dawn. In the light period, malate decarboxylation concentrates CO 2 around RuBisCO for secondary fixation. CAM mutants lacking PPC have not been described. Here, RNAi was employed to silence CAM isogene PPC1 in Kalanchoë laxiflora . Line rPPC1-B lacked PPC1 transcripts, PPC activity, dark period CO 2 fixation, and nocturnal malate accumulation. Light period stomatal closure was also perturbed, and the plants displayed reduced but detectable dark period stomatal conductance, and arrhythmia of the CAM CO 2 fixation circadian rhythm under constant light and temperature (LL) free-running conditions. By contrast, the rhythm of delayed fluorescence was enhanced in plants lacking PPC1 . Furthermore, a subset of gene transcripts within the central circadian oscillator were up-regulated and oscillated robustly. The regulation guard cell genes involved controlling stomatal movements was also altered in rPPC1-B . This provided direct evidence that altered regulatory patterns of key guard cell signaling genes are linked with the characteristic inverse pattern of stomatal opening and closing during CAM

The Kalanchoe genome provides insights into convergent evolution and building blocks of crassulacean acid metabolism

Crassulacean acid metabolism (CAM) is a water-use efficient adaptation of photosynthesis that has evolved independently many times in diverse lineages of flowering plants. We hypothesize that convergent evolution of protein sequence and temporal gene expression underpins the independent emergences of CAM from C3 photosynthesis. To test this hypothesis, we generate a de novo genome assembly and genome-wide transcript expression data for Kalanchoë fedtschenkoi, an obligate CAM species within the core eudicots with a relatively small genome (~260 Mb). Our comparative analyses identify signatures of convergence in protein sequence and re-scheduling of diel transcript expression of genes involved in nocturnal CO2 fixation, stomatal movement, heat tolerance, circadian clock, and carbohydrate metabolism in K. fedtschenkoi and other CAM species in comparison with non-CAM species. These findings provide new insights into molecular convergence and building blocks of CAM and will facilitate CAM-into-C3 photosynthesis engineering to enhance water-use efficiency in crops

Conservation and Divergence of Circadian Clock Operation in a Stress-Inducible Crassulacean Acid Metabolism Species Reveals Clock Compensation against Stress

One of the best-characterized physiological rhythms in plants is the circadian rhythm of CO(2) metabolism in Crassulacean acid metabolism (CAM) plants, which is the focus here. The central components of the plant circadian clock have been studied in detail only in Arabidopsis (Arabidopsis thaliana). Full-length cDNAs have been obtained encoding orthologs of CIRCADIAN CLOCK-ASSOCIATED1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION1 (TOC1), EARLY FLOWERING4 (ELF4), ZEITLUPE (ZTL), FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1), EARLY FLOWERING3 (ELF3), and a partial cDNA encoding GIGANTEA in the model stress-inducible CAM plant, Mesembryanthemum crystallinum (Common Ice Plant). TOC1 and LHY/CCA1 are under reciprocal circadian control in a manner similar to their regulation in Arabidopsis. ELF4, FKF1, ZTL, GIGANTEA, and ELF3 are under circadian control in C(3) and CAM leaves. ELF4 transcripts peak in the evening and are unaffected by CAM induction. FKF1 shows an abrupt transcript peak 3 h before subjective dusk. ELF3 transcripts appear in the evening, consistent with their role in gating light input to the circadian clock. Intriguingly, ZTL transcripts do not oscillate in Arabidopsis, but do in M. crystallinum. The transcript abundance of the clock-associated genes in M. crystallinum is largely unaffected by development and salt stress, revealing compensation of the central circadian clock against development and abiotic stress in addition to the well-known temperature compensation. Importantly, the clock in M. crystallinum is very similar to that in Arabidopsis, indicating that such a clock could control CAM without requiring additional components of the central oscillator or a novel CAM oscillator

Data from: Adaptive evolution of C4 photosynthesis through recurrent lateral gene transfer

C4 photosynthesis is a complex trait that confers higher productivity under warm and arid conditions. It has evolved more than 60 times via the co-option of genes present in C3 ancestors followed by alteration of the patterns and levels of expression, and adaptive changes in the coding sequences, but the evolutionary path to C4 photosynthesis is still poorly understood. The grass lineage Alloteropsis offers unparalleled opportunities for studying C4 evolution, because it includes a C3 taxon and five C4 species that vary significantly in C4 anatomy and biochemistry. Using phylogenetic analyses of nuclear genes and leaf transcriptomes, we show that fundamental elements of the C4 pathway in the grass lineage Alloteropsis were acquired via a minimum of four independent lateral gene transfers from C4 taxa that diverged from this group more than 20 million years ago. The transfer of genes that were already fully adapted for C4 function has occurred periodically over at least the last 10 million years and has been a recurrent source for the optimization of the C4 pathway. This report shows that plant-plant lateral nuclear gene transfers can be a potent source of genetic novelty and adaptation in flowering plants